The receptor for advanced glycation end products (RAGE) plays a crucial role in several disease processes, such as diabetes, inflammation, and neurodegeneration. In this article we report multiple roles of RAGE in neuronal differentiation and neurite outgrowth. In retinoic-induced P19 embryonic carcinoma stem cells, silencing the expression of RAGE by RNA interference (RNAi) blocked differentiation of the P19 cells into neuronal cells and enhanced the formation of vimentin-positive fibroblast-like cells. RAGE knockdown inhibited retinoic acid-induced activation and blocked nuclear translocation of NF-kappaB, suggesting RAGE regulates activation of NF-kappaB. RAGE was also shown to be involved in survival of P19 cells during retinoic acid differentiation. Additionally, knockdown of RAGE strongly inhibited neurite outgrowth in retinoic acid-differentiated P19 cells, indicating that RAGE is required for neurite outgrowth of differentiated P19 cells. Retinoic acid-treated P19 cells activated GTPases, Rac1, and Cdc42. This activation of the GTPases was inhibited in RAGE-knockdown cells. In primary cerebellar granule neurons, the knockdown of RAGE also inhibited neurite outgrowth. In these cells, overexpression of dominant-negative forms of Rac1 and Cdc42 inhibited neurite outgrowth, whereas overexpression of constitutively active forms of Rac1 and Cdc42 in RAGE-deficient neurons restored neurite outgrowth, indicating that RAGE mediated neurite outgrowth through the Rac1/Cdc42 pathway. This is the first report on the role of RAGE in cell lines and primary neurons, as determined by RNAi knockdown.